Germanium-based glass polyalkenoate cement

ABSTRACT

Disclosed herein are compositions and methods for making germanium-based glass polyalkenoate cements. Also disclosed are methods for their use as bone cements for bone augmentation procedures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/397,965 filed Oct. 30, 2014, which is a national phase entry ofPCT/IB2013/001166 filed on May 3, 2013, which claims priority to U.S.Provisional Application No. 61/643,077 filed May 4, 2012 and U.S.Provisional Application No. 61/642,444 filed May 3, 2012, which are allincorporated herein by reference.

FIELD

The disclosure relates to germanium-based glass polyalkenoate cementsuseful as bone cements, including for vertebroplasty, fracturestabilizations, and repair of skeletal implants.

BACKGROUND

Glass polyalkenoate cements (GPC) (also referred to as glass ionomercements) are frequently used in dentistry as restorative and lutingagents. GPCs are theoretically attractive for other clinical uses, suchas orthopedics, because they set with a negligible exotherm. This isimportant because materials which generate heat upon setting can lead tothermal necrosis of adjacent healthy tissue. Additionally, GPCs bondwith hydroxyapatite (present in both teeth and bones) and thus the setGPC is less likely to loosen over time. Finally, GPCs can be modified torelease therapeutically beneficial ions over time. However, conventionalGPCs are based on aluminosilicate glasses which are contraindicated inorthopedics as release of Al³⁺ in vivo leads to significant adverseeffects for the patient. Fatal aluminum-induced encephalopathy, impairedosteoblastic function and hindered bone mineralization have beenreported when using GPCs that include aluminum. GPCs free of aluminumhave been attempted but those materials were not suitable as they settoo quickly and did not provide sufficient handling time prior tosetting to be able to deploy them. Some materials that did have longerhandling times before setting had lower mechanical strength and thuswere unsuitable for that reason.

What is needed are new GPCs for orthopedic applications that do notrelease aluminum ions but whose characteristics allow sufficient time tohandle the material prior to setting and deliver sufficient mechanicalstrength.

SUMMARY

Novel germanium GPCs provide working times between 5 and 10 minutes,setting times between 14 and 36 minutes, and compression strengths inexcess of 30 MPa for the first 30 days. These handling characteristicsand mechanical properties make these GPCs clinically viable asinjectable bone cements and are achieved without the use of aluminum.

In a first aspect, disclosed herein is a composition comprising a glasspowder, which comprises 0.1-0.75 mole fraction GeO₂; 0.11-0.53 molefraction ZnO; and 0.01-0.2 mole fraction CaO.

In some embodiments, the composition further comprise 0.025-0.12 molefraction SrO. In some embodiments, the compositions further comprise0.005-0.08 mole fraction each of ZrO₂ and Na₂O. In some embodiments, thecompositions comprise 0.1-0.75 mole fraction GeO₂ and 0.005-0.04 molefraction each of ZrO₂ and Na₂O. In some embodiments, the compositionsfurther comprise 0.02-0.48 mole fraction SiO₂. In some embodiments, thecompositions comprise 0.1-0.75, 0.1-0.6, 0.2-0.5 or 0.35-0.50 molefraction GeO₂. In some embodiments, the compositions comprise about 0.36mole fraction ZnO. In some embodiments, the compositions comprise0.2-0.48, 0.02-0.25 or 0.02-0.2 mole fraction SiO₂. In some embodiments,the compositions comprise about 0.04 mole fraction SrO. In someembodiments, the compositions comprise 0.01-0.35, 0.02-0.16, 0.02-0.12,0.05-0.15, or 0.07-0.13 mole fraction CaO.

In some embodiments, the compositions comprise 0.005-0.06, 0.01-0.055 or0.02-0.04 mole fraction each of ZrO₂ and Na₂O. In some embodiments, thecompositions comprise no more than 0.01 mole fraction aluminosilicates.In some embodiments, the compositions are substantially free ofaluminosilicates. In some embodiments, the compositions comprise an aciddegradable powder. In some embodiments, the compositions are radioopaque. In some embodiments, the compositions comprise a polyalkenoatecement having a glass phase made from the glass powder.

In a second aspect, disclosed herein is a method of preparing a bonecement comprising mixing the glass powder described above with anaqueous solution of a about 40%-60% by weight polyalkenoic acid in aratio of about 2:1 to 1:1, and wherein the polyalkenoic acid has aweight average molecular weight (M_(W)) of about 1,150 to 1,500,000;1,150 to 383,000; 1,150 to 114,000; or 1,150 to 22,700.

In some embodiments of the method, the aqueous solution of polyalkenoicacid is 50% by weight. In some embodiments of the method, thepolyalkenoic acid has a weight average molecular weight (M_(W)) of about12,700. In some embodiments of the method, the polyalkenoic acidcomprises polyacrylic acid.

In a third aspect, disclosed herein is a composition comprising a glasspowder, which comprises: 0 mole fraction SiO₂, 0.480 mole fraction GeO₂,0.001 combined mole fraction ZrO₂/Na₂O, and 0.119 mole fraction CaO; or0.012 mole fraction SiO₂, 0.468 mole fraction GeO₂, 0.017 combined molefraction ZrO₂/Na₂O, and 0.103 mole fraction CaO; or 0.057 mole fractionSiO₂, 0.381 mole fraction GeO₂, 0.047 combined mole fraction ZrO₂/Na₂O,and 0.115 mole fraction CaO; or 0.130 mole fraction SiO₂, 0.350 molefraction GeO₂, 0.029 combined mole fraction ZrO₂/Na₂O, and 0.091 molefraction CaO; or 0.021 mole fraction SiO₂, 0.459 mole fraction GeO₂,0.019 combined mole fraction ZrO₂/Na₂O, and 0.101 mole fraction CaO; or0.215 mole fraction SiO₂, 0.215 mole fraction GeO₂, 0.050 combined molefraction ZrO₂/Na₂O, and 0.120 mole fraction CaO; or 0 mole fractionSiO₂, 0.480 mole fraction GeO₂, 0.100 combined mole fraction ZrO₂/Na₂O,and 0.020 mole fraction CaO; and further comprises zinc and strontiumcomponents.

In some embodiments of the composition, the zinc and strontiumcomponents comprise 0.36 mole fraction ZnO and 0.04 mole fraction SrO.In some embodiments of the composition, the combined mole fractionZrO₂/Na₂O is made from equal mole fractions of ZrO₂ and Na₂O.

In a fourth aspect, disclosed herein is a composition comprising a glasspowder, which comprises 0.318 mole fraction SiO₂, 0.162 mole fractionGeO₂, 0.032 combined mole fraction ZrO₂/Na₂O, and 0.088 mole fractionCaO; and further comprising zinc and strontium components.

In some embodiments of the composition, the zinc and strontiumcomponents comprise 0.36 mole fraction ZnO and 0.04 mole fraction SrO.In some embodiments of the composition, the combined mole fractionZrO₂/Na₂O is made from equal mole fractions of ZrO₂ and Na₂O.

In a fifth aspect, disclosed herein is a kit for use in preparing a bonecement comprising the glass powders described and instructions forpreparing the bone cement.

In some embodiments, the kit further comprises a polyalkenoic acid. Insome embodiments of the kit, the polyalkenoic acid is in the form of apowder. In some embodiments of the kit, the polyalkenoic acid is in theform of an aqueous solution of a about 40%-60% by weight polyalkenoicacid in a ratio of about 2:1 to 1:1, and wherein the polyalkenoic acidhas a weight average molecular weight (M_(W)) of about 1,150 to1,500,000; 1,150 to 383,000; 1,150 to 114,000; or 1,150 to 22,700. Insome embodiments of the kit, the aqueous solution of polyalkenoic acidis 50% by weight. In some embodiments of the kit, the polyalkenoic acidhas a weight average molecular weight (M_(W)) of about 12,700. In someembodiments of the kit, the polyalkenoic acid comprises polyacrylicacid.

In a sixth aspect, disclosed herein is a method of augmenting bone,comprising the steps of: (a) preparing a bone cement comprising mixingthe any of the glass powders described above with an aqueous solution ofa about 40%-60% by weight polyalkenoic acid in a ratio of about 2:1 to1:1, and wherein the polyalkenoic acid has a weight average molecularweight (M_(W)) of about 1,150 to 1,500,000; 1,150 to 383,000; 1,150 to114,000; or 1,150 to 22,700; (b) injecting said cement into a subject inneed thereof, thereby augmenting the bone.

In some embodiments of this method, the aqueous solution of polyalkenoicacid is 50% by weight. In some embodiments of this method, thepolyalkenoic acid has a weight average molecular weight (M_(W)) of about12,700. In some embodiments of this method, the polyalkenoic acidcomprises polyacrylic acid. In some embodiments of this method, the boneaugmenting is performed on a bone fracture. In some embodiments of thismethod, the injecting is through percutaneous cannulae into a fracturedvertebrate. In some embodiments, the method further comprises the stepof inflating a balloon tamp inserted into the bone fracture prior toinjection of said bone cement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an XRD plot of sample DG200.

FIG. 2 is an XRD plot of sample DG201.

FIG. 3 is an XRD plot of sample DG202.

FIG. 4 is an XRD plot of sample DG203.

FIG. 5 is an XRD plot of sample DG204.

FIG. 6 is an XRD plot of sample DG205.

FIG. 7 is an XRD plot of sample DG206.

FIG. 8 is an XRD plot of sample DG207.

FIG. 9 is an XRD plot of sample DG208.

FIG. 10 is an XRD plot of sample DG209.

FIG. 11 is an XRD plot of sample DG210.

FIG. 12 is an XRD plot of sample DG211.

FIG. 13 illustrates the results of working time experiments on GICsamples.

FIG. 14 illustrates the results of setting time experiments on GPCsamples.

FIG. 15 illustrates maximum temperatures and exotherm profiles of GPCsamples.

FIGS. 16A and B are 3D and 2D, respectively, contour plots illustratingthe effect of varying glass composition on setting time when varyingSiO₂, CaO and GeO₂ compositions.

FIGS. 17A and B are 3D and 2D, respectively, contour plots illustratingthe effect of varying glass composition on working time when varyingSiO₂, CaO and GeO₂ compositions.

FIGS. 18A and B are 3D and 2D, respectively, contour plots illustratingthe effect of varying glass composition on setting time when varyingSiO₂, CaO and ZrO₂/Na₂O compositions.

FIGS. 19A and B are 3D and 2D, respectively, contour plots illustratingthe effect of varying glass composition on working time when varyingSiO₂, CaO and ZrO₂/Na₂O compositions.

FIG. 20 illustrates results of radiopacity testing of GPC samples.

FIG. 21 illustrates an experimental set-up for compression testing ofGPC samples.

FIG. 22 illustrates compression strength experiments of GPC samplesafter 1, 7, 30, and 180 days.

FIG. 23 illustrates an experimental set-up for biaxial flexural andbiaxial flexural modulus testing.

FIG. 24 illustrates the results of biaxial flexural strength experimentsof GPC samples for 1-180 days.

FIG. 25 illustrates biaxial flexural modulus data at 1-180 days.

FIG. 26 illustrates a finite element model of a single augmentedvertebra.

FIG. 27 illustrates solved finite element models for none augmentedcontrol, and augmented models with Zn-GPC, Simplex, Cortoss, Spineplex,DG202, DG205 and DG208.

FIG. 28 illustrates average von Mises stress in cortical shell of thefinite element model.

FIG. 29 illustrates average von Mises stress in trabecular bone in thefinite element model.

FIG. 30 illustrates average von Mises stress in the cement implant inthe finite element model.

FIG. 31 illustrates MTT assay data.

FIGS. 32A-C illustrates cell viability for 1, 7, and 30 day cementextracts.

FIGS. 33A-D provide degradation data after 1, 7, and 30 days for cementextracts for Zn, Ge, Zr and Sr.

FIGS. 34A-C provide degradation data after 1, 7, and 30 days for glassextracts for Ge, Sr and Zn.

DETAILED DESCRIPTION

A GPC is a multi-component system typically comprising a glass powdercomponent, a powder of a polyalkenoic acid, such as polyacrylic acid,and water. When all three components are mixed together, the acidattacks the glass network to release metal cations, which in turncross-link the polymeric chains of the acid to form cement comprisingreacted and unreacted glass particles embedded in a polysalt matrix.

The glass powder component is a glass ceramic and the components of thatglass powder make up a network, which can be amorphous or crystalline.In some embodiments, the glass network is substantially amorphous andmay comprise some crystallinity.

Surprisingly, it was determined that when germanium is added to the GPC,it may not merely isomorphically replace silicon in the glass network.In fact, GPC's including germanium provide a more consistent settingreaction—working times between 5 and 10 minutes, and setting time inbetween 14 and 36 min. Glasses including only silicon and no germanium,have working times between 22 seconds to 7 minutes and setting timesfrom 1 minute to no setting. Thus, the disclosed germanium-based GPC'sprovide working and setting times within the range of clinicalpracticality and balanced with reasonable strength.

Glass Component of GPC

The glass component for the GPC include one or more of: zinc, strontium,calcium, zirconium, sodium, silicon and germanium. In some embodiments,the glass component of disclosed GPCs comprise one or more of: ZnO, SrO,SiO₂, GeO₂, ZrO₂, Na₂O and CaO. ZnO and SrO act as network modifyingcomponents in the glass component. In some embodiments, the glasscomponent and the GPC is substantially free of aluminosilicates andaluminum. In some embodiments, the glass component comprises no morethan 0.01 mole fraction of aluminosilicates. In some embodiments noaluminum-containing ingredients are used in the preparation of the GPC.

In some embodiments, the glass component comprises mole fractions ofGeO₂ in the ranges of 0-0.75, 0.1-0.75, 0.1-0.6, 0.2-0.5 or 0.35-0.50.

In some embodiments, the glass component comprises a mole fraction ofZnO 0.11-0.53 or 0.35-0.37. In some embodiments, the glass componentcomprises about 0.36 mole fraction ZnO.

In some embodiments, the glass component comprises mole fractions ofSiO₂ in the ranges of 0-0.48, 0.2-0.48, 0-0.25 or 0-0.20.

In some embodiments, the glass component comprises mole fractions of SrOin the range of 0.025 to 0.12. In other embodiments, the glass componentcomprises about 0.04 mole fraction SrO.

In some embodiments, the glass component comprises mole fractions of CaOin the ranges of 0.01-0.35, 0.02-0.16, 0.02-0.12, 0.05-0.15, or0.07-0.13.

In some embodiments, the glass component comprises mole fractions ofZrO₂ in the ranges of 0-0.08, 0.005-0.06, 0.01-0.055 or 0.02-0.04.

In some embodiments, the glass component comprises mole fractions ofNa₂O in the ranges of 0-0.08, 0.005-0.06, 0.01-0.055 or 0.02-0.04.

In some embodiments the mole fraction of ZrO₂ and the mole fraction Na₂Oare the same. This provides charge compensation.

The glass component of the GPC is prepared as a powder by mixing thedesired ratio of ingredients in a mechanical mixer and packed into acrucible and fired for an hour at between 1480° C. and 1520° C. Themolten glass is then quenched in deionized (DI) water at roomtemperature and dried overnight. The glass frit is ground to provide thedesired glass powder. The glass powder is sieved to provide particlesizes of less than 45 μm.

Glass synthesis commenced with the successful melt of the Zn-glassdescribed in U.S. Pat. No. 7,981,972 (see reference 1). The molefractions of this composition is:

0.48SiO₂,0.36ZnO,0.12CaO,0.04SrO.

Germanium (GeO₂), and zirconium (ZrO₂) were substituted, individually,into the Zn-based glass for silica and calcium respectively, in a seriesof experimental melts to determine the substitution limits for bothsubstances. Germanium successfully produced a glass over a range of0-0.48 mole fractions, although no compositions with GeO₂ levels greaterthan 0.48 mole fraction were attempted. ZrO₂ successfully produced aglass when incorporated between 0-0.05 mole fractions, but failed toproduce a glass at 0.08 mole fraction. ZrO₂ was matched on a molefraction basis with Na₂O for charge compensation (see references 2 and3). The original glass having mole fractions 0.48SiO₂, 0.36ZnO, 0.12CaOand 0.04SrO was tested in comparison to the compositions of the presentdisclosure and is referred to as DG200.

Preparation of GPC

To prepare the GPC, the glass powder is mixed with an aqueous solutionof a polyalkenoic acid in a ratio of about 2:1, to about 1:1. In someembodiments, the polyalkenoic acid solution can be 40-60% by weightpolyalkenoic acid powder and distilled water. In other embodiments, thepolyalkenoic acid solution is 50%, by weight, polyalkenoic acid powderand distilled water. In some embodiments, the polykenoic acid can havean average molecular of 1,150 to 1,500,000. In other embodiments, thepolykenoic acid can have an average molecular of 1,150 to 383,000; 1,150to 114,000; 1,150 to 22,700. In one embodiment, the polyalkenoic acidpowder has a weight average molecular weight (M_(W)) of 12,700.

EXAMPLES Example 1 Generation and Application of Mathematical ModelsUsing a Design of Experiments (DoE) Approach

To estimate the coefficients of a second order canonical Scheffépolynomial, a quadratic user-defined design with twelve experimentsrepresenting different compositional variants (design points) within adefined domain (design space) was constructed using Design-Expert 8.0.4software (Stat-Ease, Inc.). These design points were determined based onthe constrained ranges for each composition: with six experiments set atthe extreme vertices (V); a further five investigating axialplane-centroids (A-CB) and one overall centroid (C) within the defineddesign space. These points are in clear agreement with Scheffé'sproposal that the interesting points of a domain are at its tops, at themiddle of the sides, at the middle of the faces and its centre ofgravity (Table 1). With the mixture design method, an equation isobtained. This formula connects Y, (ie. Response), with the fourcompositional factors (SiO₂, GeO₂, ZrO/NaO and CaO, noted respectivelyas X₁, X₂, X₃ and X₄).

The Scheffé quadratic polynomial equation fitted for working time,setting time, exotherm, compression strength, biaxial flexural strengthand modulus responses is:

Y=β ₁ X ₁+β₂ X ₂+β₃ X ₃+β₄ X ₄+β₁₂ X ₁ X ₂+β₁₃ X ₁ X ₃+β₁₄ X ₁ X ₄+β₂₃ X₂ X ₃+β₂₄ X ₂ X ₄ +e

where X₁ to X₄ represent the compositional factors, β₁₋₄ coefficientsrepresent the effect of the individual compositional factors X₁₋₄;β₁₂₋₂₄, are the coefficients of regression which represent the effectsof two-way interactions between the compositional factors and e is theresidual. From the estimated coefficients of a quadratic model presentedin pseudo and actual values, the effect of each component can bederived. Mixture experiment models were developed relating the responsevariables to proportions of pseudo-components. Pseudo-componentproportions (z_(i)) are calculated as:

z _(i)=(x _(i) −L _(i))/(1−ΣL)

where x_(i) stands for the original component proportions, L_(i) standsfor the lower bound constraint (limit) for the i^(th) component, Lstands for the sum of all lower bound constraints (limits) for allcomponents in the design, and 1 represents the mixture total. Thepseudo-components are combinations of the original (actual) components,which rescale the constrained composition region so that the minimumallowable proportion of each pseudo-component is zero. Thistransformation may provide for more precisely estimating modelcoefficients compared to using the actual component system, and as suchthe coefficients derived based on the pseudo-component scaling isreferred to in the context of the discussion to follow. Model validity,in terms of experimental versus calculated data points and graphicalrepresentation (contour plots) however, is presented in terms of actualcomponent coding. When several response characteristics y₁, y₂, . . . ,y_(n) have been modeled in the proportions of the same set of qcomponents, the desirability function approach was implemented toidentify where in the compositional design space the best overall set ofproperties (such as working time, compression strength and biaxialflexural strength and modulus) may be obtained.

TABLE 1 Design of Mixtures Compositions ZnO SrO SiO₂ GeO₂ ZrO₂ Na₂O CaODG200 0.36 0.04 0.48 0 0 0 0.12 DG201 0.36 0.04 0 0.447 0.0335 0.03350.087 DG202 0.36 0.04 0 0.48 0 0 0.12 DG203 0.36 0.04 0.215 0.215 0.050.05 0.07 DG204 0.36 0.04 0.48 0 0.05 0.05 0.02 DG205 0.36 0.04 0 0.380.05 0.05 0.12 DG206 0.36 0.04 0.447 0 0.0335 0.0335 0.087 DG207 0.360.04 0.38 0 0.05 0.05 0.12 DG208 0.36 0.04 0 0.48 0.05 0.05 0.02 DC2090.36 0.04 0.215 0.215 0.025 0.025 0.12 DG210 0.36 0.04 0.223 0.2230.0335 0.0335 0.087 DG211 0.36 0.04 0.24 0.24 0.025 0.025 0.07

Example 2 Glass Production

One half mole of each component was weighed out using an analyticalbalance (ABJ 120-4 m, Kern & Sohn GmbH, Germany) using analytical gradesof zinc oxide, strontium carbonate, silica, germanium oxide, zirconia,sodium carbonate, and calcium carbonate (Sigma-Aldrich, Oakville, CAN).For each composition in Table 1, amounts for each component was weighedto arrive at the desired ratio. Powder compositions were mixed in amechanical mixer (Twin shell dry blender, Patterson-Kelly, USA) for 1hour and then dried in an oven at 100° C. for 1 hour. Compositions werethan packed into 50 mL platinum crucibles (Alpha Aesar, USA) and firedbetween 1480° C. and 1520° C. for 1 hour in a high temperature furnace(Carbolite RHF 1600, UK). Molten glass was removed was removed andquenched in deionized water at room temperature and dried overnight inan oven at 100° C. The resulting glass frit was ground using a planetaryball mill (Pulverisette 7, Fritsch GmbH, Germany) and sieved(Cole-Palmer, Montreal, Canada) to retrieve glass powder with particlesize less than 45 μm.

The glasses made include those without germanium for comparison andmodeling purposes. This method produced both germanium and non-germaniumglasses (table 1), that are representative examples of the fullcompositional space bounded by the aforementioned constraints. Theseexamples compositions can be evaluated to ascertain specificcontributions of SiO₂, GeO₂, ZrO₂/Na₂O, and CaO with regards to theproperties of interest.

Example 3 Characterization of Glasses

Differential Scanning Calorimetry (DSC)

All powders were analyzed with DSC (Q200 DSC, TA Instruments, Grimsby,ON) to determine the glass transition temperature (T_(g)). This processinvolved 45 to 50 mg of powder placed into stainless steel closed pans,while the reference pan was left empty. Samples were heated at a rate of10° C./min to a maximum temperature of 725° C. Q Series software (TAInstruments, Grimsby, CAN) was used to determine T_(g), the temperaturecorresponding the point of inflection between two user-identifiedplateaus before and after the endothermic glass transition event. Themeasured T_(g) are shown below in Table 2:

TABLE 2 Glass Transition Temperatures Composition T_(g) [° C.] DG200676.4 DG201 593 DG202 605.2 DG203 612.7 DG204 656.93 DG205 601.28 DG206644.53 DG207 640.24 DG208 581.76 DG209 624.17 DG210 612.23 DG211 621.87

X-Ray Diffraction (XRD)

The 12 glasses were analyzed with X-ray diffraction (XRD) to determinewhether the glasses were amorphous materials. XRD measurements for theparticles were performed using an INEL CPS-120 diffractometer with acurved position sensitive detector coupled to an X-ray generator (40 kV;35 mA) and equipped with a copper (Cu) target X-ray tube. Samples wereprepared by pressing the glass powders into hollow square steel wafers.A monochromator in the incident beam path limits the wavelengthsstriking the sample to Cu Kα1,α2. The X-ray beam is incident upon thesample at approximately 6′ and the curved position sensitive detectorcollects all scattered X-rays in the scan angle range 10°<2θ<100°. Theresults confirmed that the glasses were indeed amorphous. The XRD plotsare provided in FIGS. 1-12.

Example 4 Glass Annealing

Glasses were annealed to relieve internal stresses within the glassnetwork and improve handling characteristics as described by Neve, etal. The 12 glasses were annealed in the furnace at temperatures 30° C.below their respective glass transition temperatures. Clean platinumcrucibles were loosely filled with glass powders and placed in thefurnace once the furnace temperature achieved steady state. Temperatureswere monitored using a high temperature type-K thermocouple (Omega,Laval, CAN), connected to a digital thermometer (Omega, Laval, CAN).Samples were left at temperature for 3 hours, after which the furnacewas turned off and the samples were left to cool in the furnaceovernight. Annealed glass samples were removed and transferred to 20 mLdisposable glass vials and placed in a desiccator at room temperaturesfor storage.

Example 5 Cement Preparation

Cements were prepared by mixing glass power with aqueous solution ofpolyacylic acid (PAA); M_(w)=12,700 (E6, Advanced Healthcare Limited,Kent, UK) on dental mixing pads using a dental spatula. Throughout allexperiments the powder to liquid ratio was set at 2:1.5, a ratioconsistent with the literature of GPCs for vertebroplasty (as shown inreferences 5-7). The PAA solution was a 50%, by weight, PAA powder anddistilled water. All weights and volumes were measured to ±0.001 g and±0.001 mL respectively.

Example 6 Determination of Working Time (WT) for Cements

The working time of the cement was determined in accordance with theprocedure set out in ISO9917—Dentistry—Water-based cements (seereference 8). Working time is defined as the “period of time, measuredfrom the start of mixing, during which it is possible to manipulate adental material without an adverse effect on its properties.”Appropriate amounts of glass powder and PAA liquid were measured out ona dental mixing pad to make up 0.4 g of cement. The timer was startedand components were thoroughly mixed by hand using a dental spatulauntil a homogenous solution was achieved, with no visual signs of PAApowder. The liquid cement was worked with the dental spatula until itthickened to a viscosity similar to that of chewing gum, at which pointthe timer was stopped. Each of the 12 cements were tested 3 times, theaverage of which was recorded as the working time. The results are setout in Table 3 and FIG. 13. Clinically useful working times are between5 and 10 minutes. As can be seen DG200, the predicate aluminum-free GPC,is well short of this range and thus impractical for clinical use.

Example 7 Determination of Setting Time (ST) for Cements

Setting times were also evaluated following ISO9917. The apparatus usedfor this procedure are listed as follows:

-   -   8 mm×10 mm×5 mm aluminum mold, with sides covered in a thin        layer of petroleum jelly    -   75 mm×100 mm×8 mm aluminum plate wrapped in aluminum foil    -   Cabinet maintained at a temperature of (37±1)° C.    -   Gilmore needle with a mass of 453 g and a flat indenter tip with        Ø 1.1 mm    -   ×2 magnifying lens

One gram of cement was prepared and loaded into the mold, placed on thealuminum plate. At the end of mixing the timer was started. Sixtyseconds after the end of the mixing the assembly was placed in thecabinet. Sixty seconds prior to the material's working time, theassembly was gently raised upwards such that the cement's surface waspressed into the tip of the indenter. This process was repeatedintermittently until the cement could take the full weight of theindenter for 5 s, whilst making a full circular indentation in thecement. The indentation process then continued every 30 s until theindenter tip failed to make a complete circular impression in thecement's surface when viewed at 2× magnification. The timer was stopped,and the elapsed time recorded. This process was repeated twice more withthe indentation process starting 3 min before the previous recordedtime. Each cement composition was tested three times and the settingtime was taken as the average. The results are shown in Table 3 and FIG.14. DG 204 did not set. Useful setting times in a clinical settingdepend on the application. For some applications, around 18 minutes is auseful setting time.

TABLE 3 Design WT ST EX Sample Points (sec) (sec) (° C.) DG 200 V 77 12541 DG 201 V 318 838 30 DG 202 V 358 967 31 DG 203 V 425 6259 27 DG 204 V428 n/a 27 DG 205 V 298 848 31 DG 206 A-CB 69 196 36 DG 207 A-CB 22 6343 DG 208 A-CB 602 2155 27 DG 209 A-CB 302 854 30 DG 210 A-CB 416 216529 DG 211 C 474 4248 28

Example 8 Determination of Setting Exotherm (EX) for Cements

Three T-type thermocouples (Omega, Laval, CAN) were used with referencejunctions in ice water at 0° C. Two thermocouples were used to measurecement temperatures, while the third was used to measure the ambienttemperature. Alligator clips joined the thermocouple leads to BNCconnectors that fed into a BNC connector board (BNC-2120, NationalInstruments, Vaudreuil-Dorion, CAN), connected to a NI-PCI-6035 dataacquisition card (National Instruments, Vaudreuil-Dorion, CAN). LabView9.0 (National Instruments, Vaudreuil-Dorion, CAN) was used to constructa program to obtain the voltages of the three thermocouples, calculatetemperatures, graphically and numerically display the temperatures inreal time, and record the data of the two cement thermocouples toseparate text files. The program sampled data at a rate of 5000 Hz,recording 500 samples at a time. The mean voltage (V) of these sampleswas found, and temperature (T) calculated in degrees Celsius accordingto the T-type thermocouple equation:

T=a ₀ +a ₁ V+a ₂ V ² +a ₃ V ³ +a ₄ V ⁴ +a ₅ V ⁵ +a ₆ V ⁶ +a ₇ V ⁷

wherein,

a₀=0.100860910

a₁=25727.94369

a₂=−767345.8295

a₃=78025595.81

a₄=−9247486589

a₅=6.97688 E+11

a₆=−2.66192 E+13

a₇=3.94078 E+14

This process was repeated every 0.1 s until the user stopped the datacollection.

To validate the system, two test procedures were performed. First, toinvestigate the accuracy of the system, the three thermocouples wereplaced a beaker of boiling water and temperatures were recorded untiltemperature stabilized at the boiling point. These temperatures werecompared against the temperature measurements of a calibrated digitalthermometer of known accuracy (HH508 with K-type thermocouple, Omega,Laval, CAN).

One gram of cement was prepared and loaded into a plastic mold (Ø 15mm×10 mm). The thermocouples were inserted into the center of the bolusof cement and left there until the temperature peaked and decreased bymore than 1° C. from the maximum temperature. Data was plotted andanalyzed using Python 2.6.6.2 (Python Software Foundation,www.python.org). This process was conducted three times for each of the12 cements. The maximum temperature was taken as the highest temperatureachieved by the cement during any of the three trials. The maximumtemperature is shown in Table 3. FIG. 15 illustrates the exothermprofiles for the samples. As can be seen, the tested samples reachrelatively low maximum temperatures compared to alternative cements onthe market which reach 60-120° C. As mentioned previously, hightemperatures can lead to damage to surrounding healthy tissues and isthus undesirable.

Example 8 Statistical Analysis and Modeling Assessing Effect ofComponents on Working Time, Setting Time and Setting Exotherm

Statistical Analysis

Each experiment is performed in triplicate and analysed using Prism 5.0software (GraphPad software, Inc.) Results are expressed asmean±standard deviation of the triplicate determinations. One wayanalysis of variance (ANOVA) was carried out followed by a Tukey's posthoc test for comparisons between groups. The level of significance wasset at p<0.05. Results are shown in Tables 4-6 and FIGS. 16-19. FIGS.16A-B illustrate 3D (A) and 2D (B) contour plots show the effect ofvarying glass composition within the confines of the design space andthe resultant setting time based on the regression model. These plotsare confined to within the design space where component A SiO₂ variesfrom 0-0.48 mol fraction, component B GeO₂ varies from 0-0.48 mol.fraction, component D CaO varies from 0.02-0.12 mol. fraction, andZrO₂/Na₂O is fixed at 0.1 mol. fraction. FIGS. 17A-B illustrate thechanges in working time based on the regression model for the samecomposition variations as FIGS. 16A-B. FIGS. 18A-B illustrate 3D (A) and2D (B) contour plots show the effect of varying glass composition withinthe confines of the design space and the resultant setting time based onthe regression model. These plots are confined to within the designspace where component A SiO₂ varies from 0-0.48 mol fraction, componentB GeO₂ varies from 0-0.48 mol. fraction, component C ZrO₂/Na₂O variesfrom 0-0.10 mol. fraction, and CaO is fixed at 0.12 mol. fraction. FIGS.19A-B illustrate the changes in working time based on the regressionmodel for the same composition variations as FIGS. 18A-B.

TABLE 4A Regression Equations in Terms of L Pseudo Components and R2values and Summarized ANOVA for each Response. Summarized ANOVA ResponseRegression Model R² R² adj. R² pred. P Value F Working +386.56 * SiO₂0.9872 0.9648 0.9240 0.0013 44.06 Time +589.09 * GeO₂ (sec) +627.88 *ZrO₂/Na₂O +7462.16 * CaO +1196.78 * SiO₂ * GeO₂ −10890.71 * SiO₂ * CaO−9956.51 * GeO₂ * CaO −13144.13 * ZrO₂/Na₂O * CaO Setting +1568.67 *SiO₂ 0.9168 0.8337 0.8056 0.0099 11.02 Time +2569.36 * GeO₂ (sec)+146.00 * ZrO₂/Na₂O −6774.50 * CaO +2.797E+5 * SiO₂ * GeO₂ * ZrO₂/Na₂O−1.262E+5 * SiO₂ * GeO₂* CaO

TABLE 4B Working Time Regression Working Time Model for ActualComponents as well as Additional Setting Time Model and an ExothermModel Regression Model R² R² adj. R² pred. Working Time Actual+885.11020 * SiO₂ +1194.26973 * GeO₂ +1435.14798 * ZrO₂/Na₂O+12436.93684 * CaO +3557.61541 * SiO₂ * GeO₂ −32374.29770 * SiO₂ * CaO−29597.23618 * GeO₂ * CaO −39072.91707 * ZrO₂/Na₂O * CaO Setting Time L-+9.03 * SiO₂ 0.9985 0.9927 0.7756 (sec) pseudo +6.14 * GeO₂ +79.33 *ZrO₂/Na₂O −15.43 * CaO +12.25 * SiO₂ * GeO₂ −86.32 *SiO₂*ZrO₂/Na₂O−77.46 *GeO₂*ZrO/NaO +31.34 * GeO₂ * CaO −102.50 * ZrO₂/Na₂O * CaOSetting Time Actual +16.45604 * SiO₂ (sec) +9.61580 * GeO₂ +143.75249 *ZrO₂/Na₂O −25.72152 * CaO +36.42894 * SiO₂ * GeO₂ −256.60109*SiO₂*ZrO₂/Na₂O −230.25632 *GeO₂*ZrO/NaO +93.15426 * GeO₂ * CaO−304.68341 * ZrO₂/Na₂O * CaO Exotherm (° C.) L- +40.54 * SiO₂ 0.99500.9818 0.7104 pseudo +46.99 * GeO₂ +173.32 * ZrO₂/Na₂O +41.92 * CaO−31.26 * SiO₂ * GeO₂ −257.15 *SiO₂*ZrO₂/Na₂O −291.64 *GeO₂*ZrO/NaO−104.62 * GeO₂ * CaO +271.51 * ZrO₂/Na₂O * CaO Exotherm (° C.) Actual+67.48303 * SiO₂ +84.83687 * GeO₂ +280.27081 * ZrO₂/Na₂O −69.86346 * CaO−92.91738 * SiO₂ * GeO₂ −764.41338 *SiO₂*ZrO₂/Na₂O −866.94002*GeO₂*ZrO/NaO −311.00507 * GeO₂ * CaO +807.09738 * ZrO₂/Na₂O * CaO

TABLE 5 Abstracted ANOVA for the significant models (for working time(i), setting time (ii) and exotherm (iii)) investigated in this study.p-value Mean F Prob > Source Sum of Squares df Square Value F (i)Working Time (WT) (sec) Model 3.436E+005 7 49083.18 44.06 0.0013signifi- Linear 2.779E+005 3 92635.29 83.16 0.0005 cant Mixture AB59574.54 1 59574.54 53.48 0.0019 AD 6091.37 1 6091.37 5.47 0.0795 BD5091.16 1 5091.16 4.57 0.0993 CD 7888.91 1 7888.91 7.08 0.0563 Residual4455.99 4 1114.00 Cor Total 3.480E+0005 11 (ii) Setting Time (ST) (sec)Model 21.09 8 2.64 171.25 0.00058 signifi- Linear 10.91 3 3.64 236.150.0042 cant Mixture AB 5.43 1 5.43 352.54 0.0028 AC 0.38 1 0.382 4.560.0384 BC 0.30 1 0.30 19.77 0.0470 BD 1.19 1 1.19 77.23 0.0127 CD 0.35 10.35 22.71 0.0413 Residual 0.031 2 0.015 Cor Total 21.12 10 (iii)Exotherm (EX) (° C.) Model 325.04 8 40.63 75.10 0.0023 signifi- Linear212.07 3 70.69 130.67 0.0011 cant Mixture AB 40.63 1 40.63 75.10 0.0032AC 3.39 1 3.39 6.27 0.0874 BC 4.36 1 4.36 8.07 0.0656 BD 43.49 1 43.4980.40 0.0029 CD 3.37 1 3.37 6.22 0.0881 Residual 1.62 3 0.54 Cor Total326.67 11 WT - No AC, BC interactions ST - No AD interaction EX - No ADinteraction

TABLE 6 Summary of the significant (positive and negative) main andinteraction effects associated with the compositional factors (order ofsignificant effects: highest to lowest, ↑ represents positive effects,and ↓ represents negative effects). Working Time (sec) Setting Time(sec) Exotherm (° C.) Ranking Order - Ranking Order - Ranking Order -Effect of Effect of Effect of Coefficient Estimate Coefficient EstimateCoefficient Estimate Component Coefficient Component CoefficientComponent Coefficient ↓ ZrO/NaO * CaO −13144.13 ↓ ZrO/NaO * CaO −102.50↓ GeO * ZrO/NaO −291.64 ↓ SiO₂ * CaO −10890.71 ↓ SiO₂ * ZrO/NaO −86.32 ↑ZrO/NaO * CaO 271.51 ↓ GeO * CaO −9956.51 ↑ ZrO/NaO 79.33 ↓ SiO₂ *ZrO/NaO −257.15 ↑ CaO 7462.16 ↓ GeO * ZrO/NaO −77.46 ↑ ZrO/NaO 173.32 ↑SiO₂ * GeO 1196.78 ↑ GeO * CaO 31.34 ↓ GeO * CaO −104.62 ↑ ZrO/NaO627.88 ↓ CaO −15.43 ↑ GeO 46.99 ↑ GeO 598.09 ↑ SiO₂ * GeO 12.25 ↑ CaO41.92 ↑ SiO₂ 386.56 ↑ SiO₂ 9.03 ↑ SiO₂ 40.54 ↑ GeO 6.14 ↓ SiO₂ * GeO−31.26

Example 9 Determination of Radiopacity of Cements

Radiopacity of the 12 cements was also calculated according ISO9917 (seereference 8). Cement batches were prepared and loaded into aluminummolds (Ø14 mm×1.7 mm) and each face was covered with acetate paper andthe entire assembly was clamped and placed in an oven at 37° C. for 1hour. The radiopacity of each material was determined by irradiatinggroups of 3 samples alongside an aluminum step wedge (12 steps, 1.3 mmto 12.6 mm thick) at a distance of 400 mm under 70 kV and 7 mA, using aPhot-X II x-ray source (Belmont Equipment, Somerset, N.J.). Specimenswere exposed on Kodak Insight IO-41 dental film (Carestream Dental,Vaughan, ON). The optical density of each material and aluminum step wasfound using a QAS Densitometer (Picker International, Highland Heights,Ohio, USA). Each cements' ‘equivalent aluminum thickness’ was found bydividing the sample's thickness by the thickness of the aluminum stepwith an equivalent optical density. In instances where the density fellbetween two steps, the thicker step was taken, as per ISO 9917procedure. FIG. 20 illustrates the average of four measurements for eachsample. All samples exceed the ISO 9917 standard of 1 mm equivalentthickness of aluminum.

Example 10 In Vitro Compression Testing

Compression strength tests were conducted in accordance with ISO9917(see reference 8). Cement (0.800 g glass, 0.300 g PAA, 0.300 mL H₂O) wasmixed and loaded to excess into a stainless steel split mold with 5cylinders (Ø 4 mm×6 mm). Prior to filling, the mold was coated with asilicon mold release spray to facilitate sample removal. The filled moldwas clamped between two stainless steel plates with acetate paper toseparate the cement from the plates. The clamped assembly was placed inan oven at 37° C. for 1 hour. Upon removal from the oven the assemblywas broken down, cement flash was removed, and the ends of the sampleswere ground flat using wet 400 grit silicon carbide paper. The sampleswere removed from the molds and placed in plastic vials filled with 10mL of distilled water. Vials containing the specimens were incubated inan oven at 37° C. for 1, 7, 30 and 180 days. In total 240 samples wereproduced, 5 specimens for each of the 12 cement types for 4 differentincubation periods.

Compression testing was conducted using an Instron 3344 mechanicaltesting system (Instron, Norwood, Mass., USA) with a 2 kN load cell.Samples were removed from water and their diameters (do) were measuredusing digital calibers, taken as the average of two measurements to thenearest 0.01 mm, 90° apart. Specimens were coaxially positioned in thetest fixture between two pieces of damp filter paper (see FIG. 21).Specimens were crushed at a crosshead speed of 1 mm/min.Load-displacement data was recorded with Bluehill 2 (v2.25) software(Instron, Norwood, Mass., USA). Compression strength (σ_(c)) wascalculated by

${\sigma_{c} = \frac{4\; P}{\pi \; d_{c}}},$

where P was the maximum load at fracture (N). Compression strengthresults after 1, 7, 30 and 180 day incubation periods are shown in FIG.22. The compressive strength of comparable aluminum-free GPCs is 30 to50 MPa,

$\sigma_{c} = \frac{4\; P}{\pi \; d_{c}}$

Example 11 In Vitro Biaxial Flexural and Biaxial Flexural ModulusTesting

Cement (0.500 g glass, 0.188 g PAA, 0.188 mL H₂O) was mixed and loadedto excess into a Teflon mold (Ø 15 mm×1 mm). The filled mold was clampedbetween two stainless steel plates with acetate paper to separate thecement from the plates. The clamped assembly was placed in an oven at37° C. for 1 hour. Upon removal from the oven the assembly was brokendown, cement flash was removed, and the ends of the samples were groundflat using wet 400 grit silicon carbide paper. The samples were removedfrom the molds and placed in plastic vials filled with 10 mL ofdistilled water. Vials containing the specimens were incubated in anoven at 37° C. for 1, 7, 30 and 180 days.

Biaxial flexural testing was conducted similar to Williams et al. (seereference 9) and used an Instron 3344 mechanical testing system with a 2kN load cell, fitted with a biaxial flexural test fixture (see FIG. 23).The biaxial flexural test fixture was designed according to ISO6872 (seereference 11), and modified for use with the equations described byWilliams et al. (see reference 9), which employ point load from a ballbearing instead of a flat load from a pin. It consists of three 3 mmsteel ball bearings arranged to form a support ring (Ø 11 mm), and apiston with a 3 mm ball bearing to provide a point load. Samples wereremoved from water and their diameters (d_(f)) were measured usingdigital calibers, taken as the average of two measurements to thenearest 0.01 mm, 90° apart. Specimens were coaxially positioned in thecenter of the test fixture and a loaded at a crosshead speed of 1 mmmin⁻¹. Upon fracture, specimen fragments were removed and the thickness(t) at the fracture site was recorded. Load-displacement data wasrecorded with Bluehill 2 software. Biaxial flexural strength (σ_(f)) wascalculated using:

$\sigma_{f} = {{Pt}^{2}\left\lbrack {{\left( {1 + v} \right)\left( {{0.485{\ln \left( \frac{r}{t} \right)}} + 0.52} \right)} + 0.48} \right\rbrack}$

Where ν is the poisson's ratio of the cement and r is the radius of thesupport diameter. When ν=0.3, the equation becomes:

$\sigma_{f} = {{Pt}^{2}\left\lbrack {{0.63{\ln \left( \frac{r}{t} \right)}} + 1.156} \right\rbrack}$

The biaxial flexural strength measured at 1, 7 30 and 180 days is shownin FIG. 24. As can be seen these are similar to those of comparablealuminum-free GPCs known—6-11 MPa.

Biaxial flexural modulus (E) is calculated using a method produced byHiggs et al. (see reference 10) after 1, 7, 30 and 180 days ofincubation. The data of each test was recorded and analyzed using Python2.6.6.2 to determine the slope (S) of the load-displacement curve. Thiswas used in then to calculate the modulus as follows:

$\mspace{20mu} {E = {S\frac{B_{C}r^{2}}{t^{3}}}}$$B_{c} = {{- 0.0642} - {2.1900\mspace{14mu} m^{- 3}} + {\left( {0.5687 + {3.254\mspace{14mu} m^{- 3}}} \right)\left( {1 - v^{2}} \right)} + \left\lbrack {\left. \quad{{- 0.3793} + {11.0513{\mspace{11mu} \;}m^{- 3}} + {\left( {0.5223 - {7.8535\mspace{14mu} m^{- 3}}} \right)\left( {1 - v^{2}} \right)}} \right\rbrack \left( \frac{r}{r_{f}} \right)^{3}} \right.}$

B_(c) is the center of deflection function and r_(f) is the radius ofthe specimen. The modulus of each specimen was calculated and theaverage of which was biaxial flexural modulus of the material. The180-day test was done for DG 202, DG 205 and DG 208. As can be seen fromthe results shown in FIG. 25, the sample GPCs are stiffer than knownGPCs but comparable in stiffness to known alternative bone cements. Themodulus range of alternative bone cements is 1200 to 1600 MPa and themodulus range for known GPCs is 100 to 500 MPa.

Example 12 Finite Element (FE) Analysis

A FE model of a single vertebra under compressive load was used in thisinvestigation. The vertebral FE model was previously published by Tyndyket al. from computed tomography data, but modified for thisinvestigation. Specifically, the model was simplified to isolate the L4vertebra, consisting of the cortical bone shell, trabecular bone core,and vertebral arch complete with posterior elements (FIG. 26).

Cement augmentation was represented as a vertically orientatedbarrel-like volume, located in the center of the trabecular bone,equivalent to approximately 16% of the volume of the vertebral. Themodel was built, and post-processing was completed using AltairHyperworks 11.0 (Altair Engineering Canada Ltd., Toronto, Canada). Theboundary conditions consisted of a uniformly distributed 1000N axialcompressive force across the top surface, and the bottom surface wasfixed in all 6 degrees of freedom. These boundary conditions are used inliterature pertaining to FE investigations of VP. Material properties ofbone, and augmentation materials were taken from previously publisheddata shown below in Table 7. FIG. 27 illustrates solved finite elementmodels for none augmented control, and augmented models with Zn-GPC,Simplex, Cortoss, Spineplex, DG202, DG205 and DG208. The posteriorelements have been hidden for clarity.

TABLE 7 Element Material Properties Component Type E (MPa) v CorticalBone 8-node brick 12 000 0.3 Trabecular Bone 8-node brick 344 0.2Posterior 4-node tetra 3500 0.3 Elements Augmentation 8-node brickZn-GPC 450 0.3 Simplex P 1250 0.3 Cortoss 1350 0.3 Spineplex 1400 0.3DG202 1900 0.3 DG205 2050 0.3 DG208 1700 0.3

Verification of the mesh was completed via a convergence study of vonMises stress in a specific location, yielding a model of 20,546elements; with an average size of 1.4 mm. Tyndyk et al. experimentallyvalidated the original model, and the current model was validatedqualitatively by comparison with other models in the literature, showinggood agreement with respect to magnitude and distribution of stress.

The model was used to produce data for healthy vertebra with each of thefollowing seven implant materials; DG202, DG205, DG208, DG200, SimplexP®, Cortoss®, Spineplex® (clinically used, commercial materials, all ofStryker International), and non-augmented controls. The load scale ofeach run was adjusted to a lower limit of 0.00 MPa and an upper limit of3.00 MPa (von Mises), the minimum stress range encompassing the resultsof all solved models, allowing for qualitative comparison of stressthroughout the vertebral body between the different implant materials.Quantitative measurements were recorded for three regions: the corticalbone, the trabecular bone and the cement implant of both the healthymodel. The model was sectioned along a transverse plane at half theheight of the vertebral body and the stress of the exposed nodes acrossall three regions were recorded, averaged, and compared using ANOVAstatistical analysis where p=0.01. The results are shown in FIGS. 28-30.

The results of the finite element analysis show the stiffer the cementmaterial is, the more load is taken by the cement implant, and thecortical and trabecular bone take less. The stiffness of the DG seriescements in increase order is: DG208<DG 202<DG205. The DG seriesmaterials have modulus greater than that of the zinc-silicate GPC(Zn-GPC), which results in significantly different load distributionwithin the vertebral body. The DG series cements' modulus is alsogreater than those of the commercial materials as well (Simplex,Cortoss, and Spineplex), however, DG208 produces statistically similarloading patters in the augmented vertebra as the commercial materialSpineplex. DG202 and DG205 both produce statistically different loadingpatters compared to all three of the commercial materials. The importantpoints of this data are the DG series cements are statisticallydifferent from the materials described in U.S. Pat. No. 7,981,972, yetcomparable to current clinically used materials.

Example 12 In Vitro Biological Evaluation of Materials

Preparation of Material Extracts for In Vitro Analysis.

In vitro cytocompatibility as it pertains to each material (both DGseries glass and DG series cement) is evaluated using the MTT assay withevaluations being on the basis of indirect exposure via the use ofextracts.

Glass Extract Preparation.

0.1 grams of each glass powder measured to a precision of ±0.001 g witha Kern and Sohn GmbH analytical balance (model ABJ 120-4M) weretransferred into 14 mL BD Falcon™ round bottom polypropylene tubes. Theglasses were then vacuum autoclaved. in a Primus General Purpose SteamSterilizer (Primus Sterilizer Company, Inc., Omaha, Nebr.) for 15minutes at 121° C. Samples of each glass were prepared in triplicate foreach of three incubation time periods: 24 hours, 7 days, and 30 days. 10mL of tissue culture water (Sigma-Aldrich, lot #RNBB6914 and RNBC1419)were added aseptically to each sterilized glass sample, and the vialswere capped tightly. Sample vials were positioned upright in 16 mmNalgene® 5970 unwire test tube racks (Thermo Scientific) and incubatedat 37° C. in a Julabo SW22 Shaking Water Bath (Julabo USA, Inc.,Allentown, Pa.) with a uniaxial agitation rate of 2 Hz. At thecompletion of each incubation time period, samples were removed from thewater bath and extracts were decanted aseptically into 0.2 micron filtersyringes within a SterilGARD® III Advance class II biological safetycabinet. Filtrates were collected in sterile 14 mL polypropylene tubes,capped tightly and stored upright at 4° C. for later analysis.

Cement Extract Preparation.

Glass-ionomer cements were formed by mixing annealed glass powder with a50% by weight aqueous solution of a 25,000 dalton poly(acrylic acid) ina powder:liquid ratio of 2:1.5. Cements were spatulated into Ø 7 mm×1 mmteflon disc molds, clamped between flat aluminum plates using screwvises, and allowed to set in a 37° C. ambient temperature environmentfor one hour. Following setting, cement discs were removed from themolds, and transferred into 14 mL BD Falcon™ round bottom polypropylenetubes. 10 mL of sterile tissue culture water were added to each cementsample. The remainder of the extract preparation procedure is identicalto that used to prepare the glass extracts.

Fibroblast Cell Culture.

Immortalized mouse fibroblasts (NIH-3T3; American Type TissueCollection, Manassas, Va.) at passages 15-20 were used for experiments.The cells were grown in 75-cm tissue culture flasks in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 5% fetal calf serum(FCS; heat-inactivated at 56° C. for 60 min). Flasks were maintained ina humidified atmosphere at 37° C. and 10% CO₂. No antibiotics were usedduring routine subdivisions or for cell culture experiments to avoidaltering cell metabolism. At confluency, the media was discarded and 1.5mL of 0.25% trypsin EDTA solution (Sigma-Aldrich, USA, lot #1196474)were added to the cell culture flask then left for 5 to 10 minutes todetach the cells. 8.5 mL of DMEM-5% NCS was added to thetrypsin-EDTA-cell solution. 1 mL of this solution was transferred intosterile culture flasks; 19 mL of fresh media was added to each and thediluted cells were incubated at 37° C. for growth and later use. (Cellswere passaged weekly in this manner.) A sample of the remaining cellsolution was analyzed for cell density using a Bright-line Hemocytometer(Hauser Scientific, Horsham, Pa.). A portion of the cell solution wasdiluted with DMEM-5% NCS solution for a resultant 1×10⁴ cells per mLsolution in preparation for immediate use.

Assessment of Cell Viability (MTT Assay).

NIH-3T3 cells (200 μL) are seeded at a density of 1×10⁴ cells/mL in96-well plates (CoStar, Corning, Canada). Cell laden culture media wasused as a negative control, occupying one row of wells in each cultureplate (n=12). Cell culture media in the absence of cells provided ablank control in one column of an additional 96 well plate (n=8). Seededand blank plates were incubated at 37° C. for 24 hours. Followingincubation, 20 μL of sterile tissue culture water were added to eachcontrol well, blank and negative alike, while 20 μL of sample extractwere added to wells for cell viability testing. Each extract type wastested three times (n=3 extracts per condition) with a cell viabilityanalysis of n=7 for each individual extract. The plates were incubatedagain for 24 hours at 37° C. 15 mL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wereprepared in pH 7.4, 0.01M phosphate buffer solution (Sigma-Aldrich USA,lot #028K8214) shielded with an aluminum foil covering, and stored at 4°C. Following the second 24 hour incubation of the plates, 22 μL of thisMTT solution (an amount equivalent to 10% by volume of the well content)were added to each well. Samples were incubated for another 3 hours at37° C. Liquid contents of the plates were then blotted onto towels, and100 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich USA, lot #14196PMV)was added to each well of cells. Plates were shielded with aluminum foiland stirred on a rotating plate. Spectrophotometric optical density(absorption) values were read using a Bio-Tek™ Synergy HT plate readerequipped with KCF Kineticalc for Windows (Version 3.2, Rev. #2, BioTekInstruments, Inc.) A wavelength correction was performed at 977 and 900nm; plates were read at 492 nm. Cell viability was calculated accordingto 2.1 (adapted from ISO 10993-5) in comparison with the negativecontrol (seeded tissue culture water) which was set at 100% cellviability:

Cell viability %=100%(OD492e/OD492c)

Wherein:

OD492e is the mean value of the measured optical density of experimentalextract wells;OD492c is the mean value of the measured optical density of negativecontrol wells.FIG. 31 demonstrates the viability test of glass extracts followingincubation at 24 hours, 7 days, and 30 day, as compared with a seededcell culture water negative control set at 100% viability. All testedcements demonstrated high cell viability. FIGS. 32A-C demonstrateviability of the cement extracts after 24 hours, 7 days and 30 days.

Degradation Product Analysis.

1 mL of each of the glass and cement extracts was diluted up to 7.5 mLwith 2% (v/v) HNO₃. Calibration standards were prepared analytically inconcentrations ranging from 0.001 mg/L to 50 mg/L in 2% (v/v) HNO₃ fromstock solutions of 1000 mg/L zinc, strontium, silicon, germanium,zirconium, sodium, and calcium analytical standards (Perkin Elmer AtomicSpectroscopy Standards). Inductively coupled plasma optical emissionspectroscopy (ICP-OES) was applied using a Perkin Elmer Optima 8000optical emission spectrometer equipped with WinLab32 ICP software.Diluted extract concentrations were determined against empiricalcalibration curves for the following ions, listed along with theirrespective emission wavelengths: Zn (206.200 nm), Sr (407.771 nm), Si(251.611 nm), Ge (209.426 nm), Zr (343.823 nm), Na (589.592 nm) and Ca(317.933 nm). FIGS. 33A-D provide degradation data for Zn, Ge, Zr andSr, respectively after 1, 7 and 30 days for cement extracts. FIGS. 34A-Cprovide degradation data for Ge, Sr and Zn respectively after 1, 7 and30 days for glass extracts.

Assessment of Cell Cytotoxicity (LDH Assay).

Cell cytotoxicity can also be assessed in an LDH assay. The lactatedehydrogenase (LDH) assay is measured by a colorimetric lactatedehydrogenase (LDH) assay (TOX-7 (Product Code: 050M6079), SigmaAldrich, Canada), according to recommendations from the supplier. Theamount of LDH in the medium is proportional to the number of lysed/deadcells present; therefore, this assay can be used to estimate cell death.This assay measures membrane integrity as a function of the amount ofcytoplasmic LDH released into the medium. Briefly, assay mixture isprepared by mixing equal amounts of LDH assay substrate (Catalog Number:L2402), cofactor (Catalog Number: L2527) and dye solutions (CatalogNumber: L2277). For all cultures (70 μL), assay mixture is added to themedium in a proportion of two to one in 4×96-nontissue culture-treatedpolystyrene plates (CoStar, Corning, Canada). Each plate corresponds tomedium dilutions of 25, 50, 75 and 100%, respectively. Samples areincubated at room temperature in the dark (each plate covered with Alfoil) and through gentle rotation on a roller, the color reaction isstopped by 1 N HCl. Similar to the MTT assay, DMEM+5% FCS culture mediaplus sterile tissue culture water is used as a negative control andculture media plus cells plus sterile tissue culture water is used as apositive control. Absorbance is determined at 490 nm using amultidetection microplate reader (Synergy HT, BIO-TEK), with thebackground correction performed at 650 nm.

Statistical Analysis.

Each experiment is performed in triplicate and analysed using Prism 5.0software (GraphPad software, Inc.) Results are expressed asmean±standard deviation of the triplicate determinations. One wayanalysis of variance (ANOVA) was carried out followed by a Tukey's posthoc test for comparisons between groups. The level of significance wasset at p<0.05.

Example 13 Optimizing Cements for Minimum and Maximum Germanium Release

As shown above, zinc, zirconium and strontium are released at very lowconcentrations over all time periods. Germanium is released at higheramounts and thus having a composition optimized for both minimum andmaximum release of germanium is useful. The responses were modeled usingScheffé's equations quadratically (working time and [Ge⁴⁺]) as well ascubically (setting time). The general forms of the polynomials are shownbelow:

${Output}_{Q} = {{\sum\limits_{i = 1}^{q}\; {\beta_{i}\chi_{i}}} + {\sum\limits_{i = 1}^{q - 1}{\sum\limits_{j = {i + 1}}^{q}\; {\beta_{ij}\chi_{i}\chi_{j}}}} + e}$

wherein χ_(i) correspond to i^(th) compositional factors, q=4, β_(i)correspond to the effects of individual χ_(i), β_(ij) represent theeffect of two-way interactions between χ_(i) and e is the residual.

${Output}_{C} = {{\sum\limits_{i = 1}^{q}\; {\beta_{i}\chi_{i}}} + {\sum\limits_{i = 1}^{q - 1}{\sum\limits_{j = {i + 1}}^{q}\; {\beta_{ij}\chi_{i}\chi_{j}}}} + {\sum\limits_{i = 1}^{q - 1}{\sum\limits_{j = {i + 1}}^{q}\; {\gamma_{ij}\chi_{i}{\chi_{j}\left( {\chi_{i} - \chi_{j}} \right)}}}} + {\sum\limits_{i = 1}^{q - 2}{\sum\limits_{j = {i + 1}}^{q - 1}{\sum\limits_{k = {j + 1}}^{q}\; {\beta_{ijk}\chi_{i}\chi_{j}\chi_{l}}}}} + e}$

where γ_(ij) represent the coefficients of the cubic blending ofbinaries χ_(i)χ_(j) (χ_(i)−χ_(j)), and β_(ijk) represent thecoefficients of the cubic blending of ternaries χ_(i)χ_(j)χ_(i) Table 8provides the optimization criteria for maximizing [Ge⁴⁺] release. Theasterisks denote the importance of each criteria with more asterisksindicating higher importance.

TABLE 8 Criteria Set Working time Setting time 30 d Extract [GeO₂] 1 Inrange: 360-602 seconds In range: 900-1200 seconds Maximize ***** *** ***2 In range: 360-602 seconds In range: 900-1200 seconds Maximize andtarget: 360 seconds *** *** ***** 3 In range: 360-602 seconds In range:900-1200 seconds Maximize and target: 360 seconds and target: 900seconds *** ***** *** 4 In range: 360-602 seconds In range: 900-1200seconds Maximize and target: 450 seconds and target: 900 seconds ******** *** 5 In range: 360-602 seconds In range: 900-1200 secondsMaximize ***** and target: 900 seconds *** ***

Using the above models and criteria, cements that would optimize releaseof germanium for each of the above criteria have the following:

ZrO₂/Na₂O (combined mole Criteria set SiO₂ GeO₂ fraction) CaODesirability 1 0 0.480 0.001 0.119 1.00 2 0.012 0.468 0.017 0.103 0.9743 0.057 0.381 0.047 0.115 0.803 4 0.130 0.350 0.029 0.091 0.809 5 0.0210.459 0.019 0.101 0.948Zinc and strontium are added to each of the above combinations. In oneembodiment those additions are 0.36 mole fraction ZnO and 0.04 molefraction SrO. In some embodiments, the combined mole fraction ZrO₂/Na₂Ois achieved by providing equal mole fractions of each of ZrO₂ and Na₂O.

When criteria 5 from Table 8 is used and the germanium release isinstead to be minimized, an optimal glass has the composition:

ZrO₂/Na₂O (combined mole Criteria set SiO₂ GeO₂ fraction) CaODesirability 5 (but 0.318 0.162 0.032 0.0088 0.914 minimizing [Ge⁴⁺]Here also, zinc and strontium are added and in one embodiment it is inthe amounts of 0.36 mole fraction ZnO₂ and 0.04 mole fraction SrO₂. Insome embodiments, the combined mole fraction ZrO₂/Na₂O is achieved byproviding equal mole fractions of each of ZrO₂ and Na₂O.

Example 14 Cadaveric Study

A maximum of twenty cadaveric thoracic vertebrae are disarticulated,cleaned of soft tissue and separated into four different groups. If thesize or shapes of the posterior elements of the vertebrae prevent theloading of the specimens into the compression test fixture, than theposterior elements are removed, as others have done in the literature.Anterior, posterior, left and right lateral heights are recorded andaveraged for each specimen. Impressions are made of the superior andinferior surfaces of each vertebra using a semi-cured molding material,to ensure even distribution of compressive load. Specimens will beincubated in 37° C. water for 24 hours. Specimens are loaded into theInstron 3344 mechanical testing machine with their respective molds.Specimens are compressed at a rate of 0.5 mm/s until a 25% reduction inheight is seen. A height loss of 25% is part of the clinical definitionof a vertebral body compression fractures. For all specimens, max loadand stiffness are recorded. The max load is taken as the peak loadduring the trial, and stiffness is taken as the slope of theforce-displacement curve.

Commercial cements are prepared according to manufacturer'sinstructions. The cements disclosed herein will be prepared according toExample 5.

Augmentation is conducted on a maximum of 15 fractured specimens, 5 foreach cement type. Cement is injected through two 11-gauge bone biopsyneedles into the fractured vertebral body. The volume of cement isdetermined at the time of testing, ensuring the same volume isadministered to each specimen. Typical volumes are between 2 and 8 ml.Specimens are incubated in 37° C. water for 24 hours. Specimens areloaded into the Instron 3344 mechanical testing machine with theirrespective molds. New anterior, posterior, left and right lateralheights are recorded and averaged for each specimen. All specimens arerecompressed (even the non-augmented controls, acting as untreatedcontrols) at a rate of 0.5 mm/s until a further 25% reduction in heightis seen. A height loss of 25% is part of the clinical definition of avertebral body compression fractures. Post treatment max load is takenas the peak load during the trial. Stiffness is taken as the gradient ofthe force-displacement curve prior to failure. The results of theaugmented specimens is normalized using the initial strengths andstiffness to determine the percent change in strength and stiffness ofthe vertebral body post injection. This allows comparison of performanceof the novel cements to the commercial controls, limiting the influenceof size variation (T2 vs. T12) on the strength and stiffness comparison.

Strength and stiffness of each will be collected from all samples forthe initial compression, and compression after augmentation. This datawill be collected using an Instron 3344 Single Column Testing System,with Bluehill 2 Materials Testing Software (Instron, Norwood, Mass.,USA).

Example 15 Kits

Also provided are kits for preparing bone cement. Kits include glasspowders having the disclosed ratios of components and instructions forpreparing a cement from the glass powder.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the invention.

REFERENCES

-   1. Boyd, D., et al., Zinc-based glass polyalkenoate cements with    improved setting times and mechanical properties. Acta    biomaterialia, 2008. 4(2): p. 425-31.-   2. Rajmohan, N., P. Frugier, and S. Gin, Composition effects on    synthetic glass alteration mechanisms: Part 1. Experiments. Chemical    Geology, 2010. 279(3,Ä14): p. 106-119.-   3. Angeli, F., et al., Influence of zirconium on the structure    ofpristine and leached soda-lime borosilicate glasses: Towards a    quantitative approach by 17O MQMAS NMR. Journal of Non-Crystalline    Solids, 2008. 354(31): p. 3713-3722.-   4. Neve, A. D., V. Piddock, and E. C. Combe, The effect of glass    heat treatment on the properties of a novel polyalkenoate cement.    Clinical Materials, 1993. 12(2): p. 113-115.-   5. Boyd, D., et al., Comparison of an experimental bone cement with    surgical Simplex P, Spineplex and Cortoss. Journal of materials    science. Materials in medicine, 2008. 19(4): p. 1745-52.-   6. Clarkin, O., D. Boyd, and M. R. Towler, Strontium-based glass    polyalkenoate cements for luting applications in the skeleton.    Journal of biomaterials applications, 2010. 24(6): p. 483-502.-   7. Clarkin, O. M., D. Boyd, and M. R. Towler, Comparison of failure    mechanisms for cements used in skeletal luting applications. Journal    of materials science. Materials in medicine, 2009. 20(8): p.    1585-94.-   8. ISO9917, Dentistry—Water-based cements, 2007.-   9. Williams, J. A., R. W. Billington, and G. J. Pearson, The effect    of the disc support system on biaxial tensile strength of a glass    ionomer cement. Dental Materials, 2002. 18(5): p. 376-379.-   10. Higgs, W. A. J., et al., A simple method of determining the    modulus of orthopedic bone cement. Journal of biomedical materials    research, 2001. 58(2): p. 188-195.-   11. ISO6872, Dentistry—Ceramic materials, 2008.-   12. Tsigkou O, Jones J R, Polak J M, Stevens M M. Differentiation of    fetal osteoblasts and formation of mineralized bone nodules by 45S5    Bioglass (R) conditioned medium in the absence of osteogenic    supplements. Biomaterials. 2009; 30:3542-50.

1. (canceled)
 2. A method of augmenting bone, comprising: injecting acement into a bone of a subject, the cement comprising a mixture of: (a)an acid degradable glass powder comprising: 0.1-0.75 mole fraction GeO₂;0.11-0.53 mole fraction ZnO; and 0.02-0.48 mole fraction SiO₂; whereinthe acid degradable glass powder comprises no more than 0.01 molefraction aluminosilicates, and (b) an aqueous solution of about 40% toabout 60% by weight of a polyalkenoic acid that has a weight averagemolecular weight (Mw) of about 1,150 to about 1,500,000, wherein theacid degradable glass powder and the aqueous solution are in a ratio ofabout 2:1 to about 1:1 (w:w).
 3. The method of claim 2, furthercomprising allowing the injected cement to harden.
 4. The method ofclaim 2, wherein the SiO₂ and the GeO₂ are present in a ratio of about2:1 to about 1:3 (SiO₂:GeO₂).
 5. The method of claim 2, wherein the SiO₂and the GeO₂ are present in a ratio of about 1:1 (SiO₂:GeO₂).
 6. Themethod of claim 2, wherein the acid degradable glass powder furthercomprises 0.025-0.12 mole fraction SrO.
 7. The method of claim 2,wherein the acid degradable glass powder further comprises about 0.04mole fraction SrO.
 8. The method of claim 2, wherein the acid degradableglass powder further comprises 0.005-0.08 mole fraction each of ZrO₂ andNa₂O.
 9. The method of claim 2, wherein the acid degradable glass powderfurther comprises 0.005-0.06 mole fraction each of ZrO₂ and Na₂O. 10.The method of claim 2, wherein the acid degradable glass powder furthercomprises 0.005-0.04 mole fraction each of ZrO₂ and Na₂O.
 11. The methodof claim 2, wherein the acid degradable glass powder further comprises0.01-0.055 mole fraction each of ZrO₂ and Na₂O.
 12. The method of claim2, wherein the acid degradable glass powder further comprises 0.02-0.04mole fraction each of ZrO₂ and Na₂O.
 13. The method of claim 2, whereinthe acid degradable glass powder comprises 0.1-0.6 mole fraction GeO₂.14. The method of claim 2, wherein the acid degradable glass powdercomprises 0.2-0.5 mole fraction GeO₂.
 15. The method of claim 2, whereinthe acid degradable glass powder comprises 0.35-0.5 mole fraction GeO₂.16. The method of claim 2, wherein the acid degradable glass powdercomprises about 0.36 mole fraction ZnO.
 17. The method of claim 2,wherein the acid degradable glass powder comprises 0.02-0.25 molefraction SiO₂.
 18. The method of claim 17, wherein the acid degradableglass powder comprises 0.02-0.2 mole fraction SiO₂.
 19. The method ofclaim 2, wherein the acid degradable glass powder further comprises0.01-0.35 mole fraction CaO.
 20. The method of claim 2, wherein the aciddegradable glass powder further comprises 0.02-0.16 mole fraction CaO.21. The method of claim 2, wherein the acid degradable glass powderfurther comprises 0.02-0.12 mole fraction CaO.
 22. The method of claim2, wherein the acid degradable glass powder further comprises 0.05-0.15mole fraction CaO.
 23. The method of claim 2, wherein the aciddegradable glass powder further comprises 0.07-0.13 mole fraction CaO.24. The method of claim 2, wherein the acid degradable glass powder issubstantially free of aluminosilicates.
 25. The method according toclaim 2: wherein the glass powder comprises: 0.012 mole fraction SiO₂,0.468 mole fraction GeO₂, 0.017 combined mole fraction ZrO₂/Na₂O, and0.103 mole fraction CaO; or 0.057 mole fraction SiO₂, 0.381 molefraction GeO₂, 0.047 combined mole fraction ZrO₂/Na₂O, and 0.115 molefraction CaO; or 0.130 mole fraction SiO₂, 0.350 mole fraction GeO₂,0.029 combined mole fraction ZrO₂/Na₂O, and 0.091 mole fraction CaO; or0.021 mole fraction SiO₂, 0.459 mole fraction GeO₂, 0.019 combined molefraction ZrO₂/Na₂O, and 0.101 mole fraction CaO; or 0.318 mole fractionSiO₂, 0.162 mole fraction GeO₂, 0.032 combined mole fraction ZrO₂/Na₂O,and 0.088 mole fraction CaO; wherein the glass powder further compriseszinc and strontium components.
 26. The method according to claim 25,wherein the zinc and strontium components comprise 0.36 mole fractionZnO and 0.04 mole fraction SrO.
 27. The method according to claim 25,wherein the combined mole fraction ZrO₂/Na₂O is equal mole fractions ofZrO₂ and Na₂O.
 28. The method according to claim 2, wherein the glasspowder comprises: 0.215 mole fraction SiO₂, 0.215 mole fraction GeO₂,0.050 combined mole fraction ZrO₂/Na₂O, and 0.120 mole fraction CaO;wherein the glass powder further comprises zinc and strontiumcomponents.
 29. The method according to claim 28, wherein the zinc andstrontium components comprise 0.36 mole fraction ZnO and 0.04 molefraction SrO.
 30. The method according to claim 28, wherein the combinedmole fraction ZrO₂/Na₂O is equal mole fractions of ZrO₂ and Na₂O. 31.The method according to claim 2, wherein the polyalkenoic acid ispolyacrylic acid.
 32. The method according to claim 2, wherein thepolyalkenoic acid has a weight average molecular weight (Mw) of about1,150 to 383,000.
 33. The method according to claim 2, wherein thepolyalkenoic acid has a weight average molecular weight (Mw) of about1,150 to 114,000.
 34. The method according to claim 2, wherein thepolyalkenoic acid has a weight average molecular weight (Mw) of about1,150 to 22,700.
 35. The method according to claim 2, wherein thepolyalkenoic acid has a weight average molecular weight (Mw) of about12,700.
 36. The method according to claim 2, wherein the acid degradableglass powder and the aqueous solution are in a ratio of about 2:1.5(w:w).
 37. The method according to claim 2, wherein injecting the cementinto a bone comprises injecting the cement into a bone fracture.
 38. Themethod according to claim 37, further comprising inflating a balloontamp inserted into the bone fracture prior to injecting the cement. 39.The method according to claim 2, wherein injecting the cement into abone comprises injecting the cement through a percutaneous cannulae intoa fractured vertebra.
 40. The method according to claim 39, wherein thefractured vertebra is a collapsed fractured vertebra, and the methodfurther comprises, prior to injecting the cement through thepercutaneous cannulae: creating a cavity in the collapsed fracturedvertebra, restoring the height of the collapsed fractured vertebra, orboth.
 41. The method according to claim 40, wherein the method comprisesinflating a balloon tamp inserted into the bone fracture to create thecavity in the collapsed fractured vertebra, restore the height of thecollapsed fractured vertebra, or both.